Instrument systems for integrated sample processing

ABSTRACT

An integrated system for processing and preparing samples for analysis may include a microfluidic device including a plurality of parallel channel networks for partitioning the samples including various fluids, and connected to a plurality of inlet and outlet reservoirs, at least a portion of the fluids comprising reagents, a holder including a closeable lid hingedly coupled thereto, in which in a closed configuration, the lid secures the microfluidic device in the holder, and in an open configuration, the lid is a stand orienting the microfluidic device at a desired angle to facilitate recovery of partitions or droplets from the partitioned samples generated within the microfluidic device, and an instrument configured to receive the holder and apply a pressure differential between the plurality of inlet and outlet reservoirs to drive fluid movement within the channel networks.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable

BACKGROUND OF THE INVENTION

The field of life sciences has experienced dramatic advancement over thelast two decades. From the broad commercialization of products thatderive from recombinant DNA technology, to the simplification ofresearch, development and diagnostics, enabled by the invention anddeployment of critical research tools, such as the polymerase chainreaction, nucleic acid array technologies, robust nucleic acidsequencing technologies, and more recently, the development andcommercialization of high throughput next generation sequencingtechnologies. All of these improvements have combined to advance thefields of biological research, medicine, diagnostics, agriculturalbiotechnology, and myriad other related fields by leaps and bounds.

Many of these advances in biological analysis and manipulation requirecomplex, multi-step process workflows, as well as multiple highlydiverse unit operations, in order to achieve the desired result. Nucleicacid sequencing, for example requires multiple diverse steps in theprocess workflow (e.g., extraction, purification, amplification, librarypreparation, etc.) before any sequencing operations are performed. Eachworkflow process step and unit operation introduces the opportunity foruser intervention and its resulting variability, as well as providingopportunities for contamination, adulteration, and other environmentalevents that can impact the obtaining of accurate data, e.g., sequenceinformation.

The present disclosure describes systems and processes for integratingmultiple process workflow steps in a unified system architecture thatalso integrates simplified sample processing steps.

BRIEF SUMMARY OF THE INVENTION

Provided are integrated systems and processes for use in the preparationof samples for analysis, and particularly for the preparation of nucleicacid containing samples for sequencing analysis.

According to various embodiments of the present invention, an integratedsystem for processing and preparing samples for analysis comprises amicrofluidic device including a plurality of parallel channel networksfor partitioning the samples including various fluids, and connected toa plurality of inlet and outlet reservoirs, at least a portion of thefluids comprising reagents, a holder including a closeable lid hingedlycoupled thereto, in which in a closed configuration, the lid secures themicrofluidic device in the holder, and in an open configuration, the lidcomprises a stand orienting the microfluidic device at a desired angleto facilitate recovery of partitions or droplets from the partitionedsamples generated within the microfluidic device. The integrated systemmay further include an instrument configured to receive the holder andapply a pressure differential between the plurality of inlet and outletreservoirs to drive fluid movement within the channel networks.

In some embodiments, the desired angle at which the microfluidic deviceis oriented by the lid ranges from 20-70 degrees, 30-60 degrees, 40-50degrees.

In some embodiments, the desired angle at which the microfluidic deviceis oriented by the lid is 45 degrees.

In some embodiments, the instrument comprises a retractable traysupporting and seating the holder, and slidable into out of theinstrument, a depressible manifold assembly configured to be actuatedand lowered to the microfluidic device and to sealaby interface with theplurality of inlet and outlet reservoirs, at least one fluid drivecomponent configured to apply the pressure differential between theplurality of inlet and outlet reservoirs, and a controller configured tooperate the at least one drive fluid component to apply the pressuredifferential depending on a mode of operation or according topreprogrammed instructions.

In some embodiments, at least one of the parallel channel networkscomprises a plurality of interconnected fluid channels fluidlycommunicated at a first channel junction, at which an aqueous phasecontaining at least one of the reagents is combined with a stream of anon-aqueous fluid to partition the aqueous phase into discrete dropletswithin the non-aqueous fluid, and the partitioned samples are stored inthe outlet reservoirs for harvesting, or stored in at least one productstorage vessel.

In some embodiments, the plurality of interconnected fluid channelscomprises a microfluidic structure having intersecting fluid channelsfabricated into a monolithic component part.

In some embodiments, the integrated system further comprises a gasketcoupled to the holder and including a plurality of apertures, in whichwhen the lid is in the closed configuration, the gasket is positionedbetween the reservoirs and the manifold assembly to provide the sealableinterface, and the apertures allow pressure communication between atleast one of the outlet and the inlet reservoirs and the at least onefluid drive component.

In some embodiments, the integrated system further comprises springs tobias the manifold assembly in a raised position, and a servo motor toactuate and lower the manifold assembly.

In some embodiments, the integrated system further comprises at leastone monitoring component interfaced with at least one of the pluralityof channel networks and configured to observe and monitorcharacteristics and properties of the at least one channel network andfluids flowing therein. The at least one monitoring component isselected from the group consisting of: a temperature sensor, a pressuresensor, and a humidity sensor.

In some embodiments, the integrated system further comprises at leastone valve to control flow into a segment of at least one channel of theplurality of parallel channel networks by breaking capillary forcesacting to draw aqueous fluids into the channel at a point of widening ofthe channel segment in the valve.

In some embodiments, the at least one valve comprises a passive checkvalve.

In some embodiments, at least one of the plurality of parallel channelnetworks comprises a first channel segment fluidly coupled to a sourceof barcode reagents, a second channel segment fluidly coupled to asource of the samples, the first and second channel segments fluidlyconnected at a first channel junction, a third channel segment connectedto the first and second channel segments at the first channel junction,a fourth channel segment connected to the third channel segment at asecond channel junction and connected to a source of partitioning fluid,and a fifth channel segment fluidly coupled to the second channeljunction and connected to a channel outlet, The at least one fluiddriving system is coupled to at least one of the first, second, third,fourth, and fifth channel segments, and is configured to drive flow ofthe barcode reagents and the reagents of the sample into the firstchannel junction to form a reagent mixture in the third channel segmentand to drive flow of the reagent mixture and the partitioning fluid intothe second channel junction to form droplets of the first reactionmixture in a stream of partitioning fluid within the fifth channelsegment.

According to various embodiments of the present invention, a holderassembly comprises a holder body configured to receive a microfluidicdevice, the microfluidic device including a plurality of parallelchannel networks for partitioning various fluids, and a closeable lidhingedly coupled to the holder body. In a closed configuration, the lidsecures the microfluidic device in the holder body, and in an openconfiguration, the lid comprises a stand to orient the microfluidicdevice at a desired angle to facilitate recovery of partitions ordroplets from the partitioned fluids without spilling the fluids.

In some embodiments, the desired angle at which the microfluidic deviceis oriented by the lid ranges from 20-70 degrees, 30-60 degrees, 40-50degrees.

In some embodiments, the desired angle at which the microfluidic deviceis oriented by the lid is 45 degrees.

According to various embodiments of the present invention, a method formeasurement of parameters of fluid in samples for analysis in amicrofluidic device of an integrated system comprises positioning a linecamera in optical communication with a segment of at least one fluidchannel of the microfluidic device, imaging, by the at least one linescan camera, in a detection line across the channel segment, andprocessing, by the at least one line scan camera, images of particulateor droplet based materials of the samples as the materials pass throughthe detection line, to determine shape, size and correspondingcharacteristics of the materials, and angling the at least one linecamera and the corresponding detection line across the channel segmentto increase a resolution of resulting images across the channel segment.An angle at which the at least one line camera and the correspondingdetection line are angled across the channel segment ranges from 5-80degrees from an axis perpendicular to the channel segment.

In some embodiments, the method for measurement further comprisesoptically communicating the line camera with a post partitioning segmentof at least one fluid channel of the microfluidic device, to monitorformed partitions emanating from a partitioning junction of themicrofluidic device.

In some embodiments, the method for measurement further comprisesoptically communicating the line camera with a post partitioning segmentof at least one fluid channel of the microfluidic device, to monitorformed partitions emanating from a partitioning junction of themicrofluidic device.

In some embodiments, the method for measurement further comprisesoptically coupling at least one line scan sensor to one or more of aparticle inlet channel segment to monitor materials being brought into apartitioning junction to be co-partitioned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a first level of system architecture asfurther described herein.

FIG. 2 is an exemplary illustration of a consumable microfluidiccomponent for use in partitioning sample and other materials.

FIGS. 3A, 3B, and 3C illustrate different components of a microfluidiccontrol system.

FIG. 4 schematically illustrates the structure of an example opticaldetection system for integration into overall instrument systemsdescribed herein.

FIG. 5 schematically illustrates an alternate detection scheme for usein imaging materials within microchannels.

FIG. 6 illustrates an exemplary processing workflow, some or all ofwhich may be integrated into a unified system architecture.

FIG. 7 schematically illustrates the integration of a nucleic acid sizefragment selection component into a microfluidic partitioning component.

FIG. 8 illustrates a monitored pressure profile across a microfluidicchannel network for use in controlling fluidic flows through the channelnetwork.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to devices and systems for use inapportioning reagents and other materials into extremely large numbersof partitions in a controllable manner. In particularly preferredaspects, these devices and systems are useful in apportioning multipledifferent reagents and other materials, including for example, beads,particles and/or microcapsules into large numbers of partitions alongwith other reagents and materials. In particularly preferred aspects,the devices and systems apportion reagents and other materials intodroplets in an emulsion in which reactions may be carried out inrelative isolation from the reagents and materials included withindifferent partitions or droplets. Also included are systems that includethe above devices and systems for conducting a variety of integratedreactions and analyses using the apportioned reagents and othermaterials. Thus, the systems and processes of the present invention canbe used with any devices and any systems such as those outlined in U.S.Provisional Patent Application No. 62/075,653, the full disclosure ofwhich is expressly incorporated by reference in its entirety for allpurposes, specifically including the Figures, Legends and descriptionsof the Figures and components therein.

I. Partitioning Systems

The systems described herein include instrumentation, components, andreagents for use in partitioning materials and reagents. In preferredaspects, the systems are used in the delivery of highly complex reagentsets to discrete partitions for use in any of a variety of differentanalytical and preparative operations. The systems described herein alsooptionally include both upstream and downstream subsystems that may beintegrated with such instrument systems.

The overall architecture of these systems typically includes apartitioning component, which is schematically illustrated in FIG. 1. Asshown, the architecture 100, includes a fluidics component 102(illustrated as an interconnected fluid conduit network 104), that isinterfaced with one or more reagent and/or product fluid storagevessels, e.g., vessels 106-116. The fluidics component includes anetwork of interconnected fluid conduits through which the variousfluids are moved from their storage vessels, and brought together inorder to apportion the reagents and other materials into differentpartitions, which partitions are then directed to the product storagevessel(s), e.g., vessel 116.

The fluidics component 102 is typically interfaced with one or morefluid drive components, such as pumps 118-126, and/or optional pump 128,which apply a fluid driving force to the fluids within the vessels todrive fluid flow through the fluidic component. By way of example, thesefluid drive components may apply one or both of a positive and/ornegative pressure to the fluidic component, or to the vessels connectedthereto, to drive fluid flows through the fluid conduits. Further,although shown as multiple independent pressure sources, the pressuresources may comprise a single pressure source that applies pressurethrough a manifold to one or more of the various channel termini, or anegative pressure to a single outlet channel terminus, e.g., pump 128 atreservoir 116.

The instrument system 100 also optionally includes one or moreenvironmental control interfaces, e.g., environmental control interface130 operably coupled to the fluidic component, e.g., for maintaining thefluidic component at a desired temperature, desired humidity, desiredpressure, or otherwise imparting environmental control. A number ofadditional components may optionally be interfaced with the fluidicscomponent and/or one or more of the reagent or product storage vessels106-116, including, e.g., optical detection systems for monitoring themovement of the fluids and/or partitions through the fluidic component,and/or in the reagent and or product reservoirs, etc., additional liquidhandling components for delivering reagents and/or products to or fromtheir respective storage vessels to or from integrated subsystems, andthe like.

The instrument system also may include integrated control software orfirmware for instructing the operation of the various components of thesystem, typically programmed into a connected processor 132, which maybe integrated into the instrument itself, or maintained on a directly orwirelessly connected, but separate processor, e.g., a computer, tablet,smartphone, or the like, for controlling the operation of, and/or forobtaining data from the various subsystems and/or components of theoverall system.

II. Fluidics Component

As noted above, the fluidics component of the systems described hereinis typically configured to allocate reagents to different partitions,and particularly to create those partitions as droplets in an emulsion,e.g., an aqueous droplet in oil emulsion. In accordance with thisobjective, the fluidic component typically includes a plurality ofchannel or conduit segments that communicate at a first channel junctionat which an aqueous phase containing one or more of the reagents iscombined with a stream of a non-aqueous fluid, such as an oil like afluorinated oil, for partitioning the aqueous phase into discretedroplets within the flowing oil stream. While any of a variety offluidic configurations may be used to provide this channel junction,including, e.g., connected fluid tubing, channels, conduits or the like,in particularly preferred aspects, the fluidic component comprises amicrofluidic structure that has intersecting fluid channels fabricatedinto a monolithic component part. Examples of such microfluidicstructures have been generally described in the art for a variety ofdifferent uses, including, e.g., nucleic acid and protein separationsand analysis, cell counting and/or sorting applications, high throughputassays for, e.g., pharmaceutical candidate screening, and the like.

Typically, the microfluidics component of the system includes a set ofintersecting fluid conduits or channels that have one or more crosssectional dimensions of less than about 200 um, preferably less thanabout 100 um, with some cross sectional dimensions being less than about50 um, less than about 40 um, less than about 30 um, less than about 20um, less than about 10 um, and in some cases less than or equal to about5 um. Examples of microfluidic structures that are particularly usefulin generating partitions are described herein and in U.S. ProvisionalPatent Application No. 61/977,804, filed Apr. 4, 2014, the fulldisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

FIG. 2 shows an exemplary microfluidic channel structure for use ingenerating partitioned reagents, and particularly for use inco-partitioning two or more different reagents or materials intoindividual partitions. As shown, the microfluidic component 200 providesone or more channel network modules 250 for generating partitionedreagent compositions. As shown, the channel network module 250 includesa basic architecture that includes a first channel junction 210 linkingchannel segments 202, 204 and 206, as well as channel segment 208 thatlinks first junction 210 to second channel junction 222. Also linked tosecond junction 222 are channel segments 224, 226 and 228.

As illustrated, channel segment 202 is also fluidly coupled to reservoir230, that provides, for example, a source of additional reagents such asmicrocapsules, beads, particles or the like, optionally including one ormore encapsulated or associated reagents, suspended in an aqueoussolution. Each of channel segments 204 and 206 are similarly fluidlycoupled to reagent storage vessel or fluid reservoir 232, which mayprovide for example, a source of sample material as well as otherreagents to be partitioned along with the microcapsules. As notedpreviously, although illustrated as both channel segments 204 and 206being coupled to the same reservoir 232, these channel segments areoptionally coupled to different reservoirs for introducing differentreagents or materials to be partitioned along with the reagents fromreservoir 230.

As shown, each of channel segments 202, 204 and 206 are provided withoptional additional fluid control structures, such as passive fluidvalve 236. These valves optionally provide for controlled filling of theoverall devices by breaking the capillary forces that draw the aqueousfluids into the device at the point of widening of the channel segmentin the valve structure. Briefly, aqueous fluids are introduced firstinto the device in reservoirs 230 and 232, at which point these fluidswill be drawn by capillary action into their respective channelsegments. Upon reaching the valve structure, the widened channel willbreak the capillary forces, and fluid flow will stop until acted upon byoutside forces, e.g., positive or negative pressures, driving the fluidinto and through the valve structure. These structures are alsoparticularly useful as flow regulators for instances where beads,microcapsules or the like are included within the reagent streams, e.g.,to ensure a regularized flow of such particles into the various channeljunctions.

Also shown in channel segment 202 is a funneling structure 252, thatprovides reduced system failure due to channel clogging, and alsoprovides an efficient gathering structure for materials from reservoir230, e.g., particles, beads or microcapsules, and regulation of theirflow. As also shown, in some cases, the connection of channel segment202 with reservoir 230, as well as the junctions of one or more or allof the channel segments and their respective reservoirs, may be providedwith additional functional elements, such as filtering structures 254,e.g., pillars, posts, tortuous fluid paths, or other obstructivestructures to prevent unwanted particulate matter from entering orproceeding through the channel segments.

First junction 210 is fluidly coupled to second junction 222. Alsocoupled to channel junction 222 are channel segments 224 and 226 thatare, in turn fluidly coupled to reservoir 234, which may provide, forexample, partitioning fluid that is immiscible with the aqueous fluidsflowing from junction 210. Again, channel segments 224 and 226 areillustrated as being coupled to the same reservoir 234, although theymay be optionally coupled to different reservoirs, e.g., where eachchannel segment is desired to deliver a different composition tojunction 222, e.g., partitioning fluids having different make up,including differing reagents, or the like.

In exemplary operation, a first fluid reagent, e.g., includingmicrocapsules or other reagents, that is provided in reservoir 230 isflowed through channel segment 202 into first channel junction 210.Within junction 210, the aqueous first fluid reagent solution iscontacted with the aqueous fluids, e.g., a second reagent fluid, fromreservoir 232, as introduced by channel segments 204 and 206. Whileillustrated as two channel segments 204 and 206, it will be appreciatedthat fewer (1) or more channel segments may be connected at junction210. For example, in some cases, junction 210 may comprise a T junctionat which a single side channel meets with channel segment 202 injunction 210.

The combined aqueous fluid stream is then flowed through channel segment208 into second junction 222. Within channel junction 222, the aqueousfluid stream flowing through channel segment 208, is formed intodroplets within the immiscible partitioning fluid introduced fromchannel segments 224 and 226. In some cases, one or both of thepartitioning junctions, e.g., junction 222 and one or more of thechannel segments coupled to that junction, e.g., channel segments 208,224, 226 and 228, may be further configured to optimize the partitioningprocess at the junction.

Further, although illustrated as a cross channel intersection at whichaqueous fluids are flowed through channel segment 208 into thepartitioning junction 222 to be partitioned by the immiscible fluidsfrom channel segments 224 and 226, and flowed into channel segment 228,as described elsewhere herein, partitioning structure within amicrofluidic device of the invention may comprise a number of differentstructures.

As described in greater detail below, the flow of the combined first andsecond reagent fluids into junction 222, and optionally, the rate offlow of the other aqueous fluids and/or partitioning fluid through eachof junctions 210 and 222, are controlled to provide for a desired levelof partitioning, e.g., to control the number of frequency and size ofthe droplets formed, as well as control apportionment of othermaterials, e.g., microcapsules, beads or the like, in the droplets.

Once the reagents are allocated into separate partitions, they areflowed through channel segment 228 and into a recovery structure orzone, where they may be readily harvested. As shown, the recovery zoneincludes, e.g., product storage vessel or outlet reservoir 238.Alternatively, the recovery zone may include any of a number ofdifferent interfaces, including fluidic interfaces with tubes, wells,additional fluidic networks, or the like. In some cases, where therecovery zone comprises an outlet reservoir, the outlet reservoir willbe structured to have a volume that is greater than the expected volumeof fluids flowing into that reservoir. In its simplest sense, the outletreservoir may, in some cases, have a volume capacity that is equal to orgreater than the combined volume of the input reservoirs for the system,e.g., reservoirs 230, 232 and 234.

In certain aspects, and as alluded to above, at least one of the aqueousreagents to be co-partitioned will include a microcapsule, bead or othermicroparticle component, referred to herein as a bead. As such, one ormore channel segments may be fluidly coupled to a source of such beads.Typically, such beads will include as a part of their composition one ormore additional reagents that are associated with the bead, and as aresult, are co-partitioned along with the other reagents. In many cases,the reagents associated with the beads are releasably associated with,e.g., capable of being released from, the beads, such that they may bereleased into the partition to more freely interact with other reagentswithin the various partitions. Such release may be driven by thecontrolled application of a particular stimulus, e.g., application of athermal, chemical or mechanical stimulus. By providing reagentsassociated with the beads, one may better control the amount of suchreagents, the composition of such reagents being co-partitioned, and theinitiation of reactions through the controlled release of such reagents.

By way of example, in some cases, the beads may be provided witholigonucleotides releasably associated with the beads, where theoligonucleotides represent members of a diverse nucleic acid barcodelibrary, whereby an individual bead may include a large number ofoligonucleotides, but only a single type of barcode sequence includedamong those oligonucleotides. The barcode sequences are co-partitionedwith sample material components, e.g., nucleic acids, and used tobarcode portions of those sample components. The barcoding then allowssubsequent processing of the sequence data obtained, by matchingbarcodes as having derived from possibly structurally related sequenceportions. The use of such barcode beads is described in detail in U.S.patent application Ser. No. 14/316,318, filed Jun. 26, 2014, andincorporated herein by reference in its entirety for all purposes.

The microfluidic component is preferably provided as a replaceableconsumable component that can be readily replaced within the instrumentsystem, e.g., as shown in FIG. 2. For example, microfluidic devices orchips may be provided that include the integrated channel networksdescribed herein, and optionally include at least a portion of theapplicable reservoirs, or an interface for an attachable reservoir,reagent source or recovery component as applicable. Fabrication and useof microfluidic devices has been described for a wide range ofapplications, as noted above. Such devices may generally be fabricatedfrom organic materials, inorganic materials, or both. For example,microfluidic devices may be fabricated from organic materials, such aspolyethylene or polyethylene derivatives, such as cyclic olefincopolymers (COC), polymethylmethacrylate (PMMA), polydimethylsiloxane(PDMS), polycarbonate, polystyrene, polypropylene, or the like, or theymay be fabricated in whole or in part from inorganic materials, such assilicon, or other silica based materials, e.g., glass, quartz, fusedsilica, borosilicate glass, or the like. Particularly usefulmicrofluidic device structures and materials are described inProvisional U.S. Patent Application No. 61/977,804, filed Apr. 4, 2014,previously incorporated herein by reference.

III. Flow Controllers

As noted with reference to FIG. 1, above, typically, such replaceablemicrofluidics structures are integrated within a larger instrumentsystem that, as noted above, includes a number of other components foroperation of the system, as well as optional additional systemcomponents used for monitoring system operation, and/or for processes ina workflow that sit upstream and/or downstream of the partitioningprocesses.

In particular, as noted above, the overall system typically includes oneor more fluid driving systems for driving flow of the fluid reagentsthrough the channel structures within the fluidic component(s). Fluiddriving systems can include any of a variety of different fluid drivingmechanisms. In preferred aspects, these fluid driving systems willinclude one or more pressure sources interfaced with the channelstructures to apply a driving pressure to either push or pull fluidsthrough the channel networks. In particularly preferred aspects, thesepressure sources include one or more pumps that are interfaced with oneor more of the inlets or outlets to the various channel segments in thechannel network.

As will be appreciated, in some cases, fluids are driven through thechannel network through the application of positive pressures byapplying pressures to each of the inlet reservoirs through theinterconnected channel segments. In such cases, one or more pressuresources may be interfaced with each reservoir through an appropriatemanifold or connector structure. Alternatively, a separatelycontrollable pressure source may be applied to each of one or more ofthe various different inlet reservoirs, in order to independentlycontrol the application of pressure to different reservoirs. Suchindependent control can be useful where it is desired to adjust ormodify of flow profiles in different channel segments over time or fromone application to another. Pressure pumps, whether for application ofpositive or negative pressure or both, may include any of a variety ofpumps for application of pressure heads to fluid materials, including,for example, diaphragm pumps, simple syringe pumps, or other positivedisplacement pumps, pressure tanks or cartridges along with pressureregulator mechanisms, e.g., that are charged with a standing pressure,or the like.

As noted, in certain cases, a negative pressure source may be applied tothe outlet of the channel network, e.g., by interfacing the negativepressure source with outlet reservoir 238 shown in FIG. 2. By applying anegative pressure to the outlet, the ratios of fluid flow within all ofthe interconnected channels is generally maintained as relativelyconstant, e.g., flow within individual channels are not separatelyregulated through the applied driving force. As a result, flowcharacteristics are generally a result of one or more of the channelgeometries, e.g., cross section and length which impact fluidicresistance through such channels, fluid the properties within thevarious channel segments, e.g., viscosity, and the like. While notproviding for individual flow control within separate channel segmentsof the device, it will be appreciated that one can program flow ratesinto a channel structure through the design of the channel network,e.g., by providing varied channel dimensions to impact flow rates undera given driving force. Additionally, use of a single vacuum sourcecoupled to the outlet of the channel network provides advantages ofsimplicity in having only a single driving force applied to the system.

In alternative or additional aspects, other fluid driving mechanisms maybe employed, including for example, driving systems that are at leastpartially integrated into the fluid channels themselves, such aselectrokinetic pumping structures, mechanically actuated pumpingsystems, e.g., diaphragm pumps integrated into the fluidic structures,centrifugal fluid driving, e.g., through rotor based fluidic componentsthat drive fluid flow outward from a central reservoir through aradially extending fluidic network, by rapidly spinning the rotor, orthrough capillary force or wicking driving mechanisms.

The pump(s) are typically interfaced with the channel structures by asealed junction between the pump, or conduit or manifold connected tothe pump, and a terminus of the particular channel, e.g., through areservoir or other interfacing component. In particular, with respect tothe device illustrated in FIG. 2, a pump outlet may be interfaced withthe channel network by mating the pump outlet to the opening of thereservoir with an intervening gasket or sealing element disposed betweenthe two. The gasket may be an integral part of the microfluidicstructure, the pump outlet, or both, or it may be a separate componentthat is placed between the microfluidic structure and the pump outlet.For example, an integrated gasket element may be molded over the toplayer of the microfluidic device, e.g., as the upper surface of thereservoirs, as a second deformable material, e.g., a thermoplasticelastomer molded onto the upper lip of the reservoir that is molded fromthe same rigid material as the underlying microfluidic structure.Although described with reference to pressed interfaces of pump outletsto reservoirs on microfluidic devices, it will be appreciated that avariety of different interface components may be employed, including anyof a variety of different types of tubing couplings (e.g., barbed, quickconnect, press fit, etc.) to interface pressure sources to channelnetworks. Likewise, the pressure sources may be interfaced to upstreamor downstream process components and communicated to the channelnetworks through appropriate interface components between the fluidiccomponent in the partitioning system and the upstream or downstreamprocess component. For example, where multiple integrated components arefluidically coupled together, application of a pressure to one end ofthe integrated fluidic system may be used to drive fluids through theconduits of each integrated component as well as to drive fluids fromone component to another.

In some cases, both positive and negative pressures may be employed in asingle process run. For example, in some cases, it may be desirable toprocess a partitioning run through a microfluidic channel network. Uponconclusion of the run, it may be desirable to reverse the flow throughthe device, to drive some portion of the excess non-aqueous componentback out of the outlet reservoir back through the channel network, inorder to reduce the amount of the non-aqueous phase that will be presentin the outlet reservoir when being accessed by the user. In such cases,a pressure may be applied in one direction, either positive or negative,during the partitioning run to create the droplets through themicrofluidic device, e.g., device 200 in FIG. 2, that accumulate inreservoir 238 along with excess non-aqueous phase material, which willremain at the bottom of the reservoir, e.g., at the interface with thechannel 228. By then reversing the direction of pressure, eitherpositive or negative, one may drive excess non-aqueous material backinto the channel network, e.g., channel 228.

Additional control elements may be included coupled to the pumps of thesystem, including valves that may be integrated into manifolds, forswitching applied pressures as among different channel segments in asingle fluidic structure or between multiple channel structures inseparate fluid components. Likewise, control elements may also beintegrated into the fluidics components. For example, valving structuresmay be included within the channel network to controllably interruptflow of fluids in or through one or more channel segments. Examples ofsuch valves include the passive valves described above, as well asactive controllable valve structures, such as depressible diaphragms orcompressible channel segments, that may be actuated to restrict or stopflow through a given channel segment.

FIGS. 3A-3C illustrate components of an exemplary instrument/systemarchitecture for interfacing with microfluidic components, as describedabove. As shown in FIG. 3A, a microfluidic device 302 that includesmultiple parallel channel networks all connected to various inlet andoutlet reservoirs, e.g., reservoirs 304 and 306, is placed into asecondary holder 310 that includes a closeable lid 312, to secure thedevice within the holder. Once the lid 312 is closed over themicrofluidic device 302 in the secondary holder 310, an optional gasket314 may be placed over the top of the reservoirs, e.g., reservoirs 304and 306, protruding from the top of the secondary holder 310. As shown,gasket 314 includes apertures 316 to allow pressure communicationbetween the reservoirs, e.g., reservoirs 304 and 306, and an interfacedinstrument, through the gasket. As shown, gasket 314 also includessecuring points 318 that are able to latch onto complementary hooks orother tabs 320 on the secondary holder to secure the gasket 314 inplace. Also as shown, secondary holder 310 is assembled such that whenthe lid portion 312 is fully opened, it creates a stand for thesecondary holder 310 and a microfluidic device, e.g., microfluidicdevice 302, contained therein, retaining the microfluidic device 302 atan appropriate orientation, e.g., at a supported angle, for recoveringpartitions or droplets generated within the microfluidic device 302.Typically, the supported angle at which the microfluidic device 302 isoriented by the lid 312 will range from about 20-70 degrees, moretypically about 30-60 degrees, preferrably 40-50 degrees, or in somecases approximately 45 degrees. Though recited in terms of certainranges, it will be understood that all ranges from the lowest of thelower limits to the highest of the upper limits are included, includingall intermediate ranges or specific angles, within this full range orany specifically recited range. Such angles provide an improved oroptimized configuration for recovering the partitions or dropletsgenerated within the microfluidic device 302 while minimizing orpreventing spillage of the fluids within the microfluidic device 302.

FIG. 3B shows a perspective view of an instrument system 350 while FIG.3C illustrates a side view of the instrument system 350. As shown, andwith reference to FIG. 3A, a microfluidic device 302 may be placed intoa secondary holder 310 that is, in turn, placed upon a retractable tray322, that moves is slidable into and out of the instrument system 350.The retractable tray 322 is positioned on guide rails 324 that extend ina horizontal direction of the instrument system 350 (as shown by thearrows in FIG. 3C) and allow the retractable tray 322 to slide into andout of a slot formed in the housing 354 when driven by a drivingmechanism. In some embodiments, the driving mechanism may include amotor part (not shown) to transmit rotation power, and a moving linkpart (not shown) extending from the motor part towards the guide rails324, such that the moving link part is connected to the guide rails 324to slide the guide rails 324 in the horizontal direction when the motorpart is operated. Pinion gears (not shown) may be formed on the movinglink part and rack gears (not shown) extending in the horizontaldirection may be formed on the guide rails 324 such that the piniongears are engaged with the rack gears, and when the motor part isoperated, the moving link part is rotated and the pinion gears arerotated and moved along the rack gears to slide the retractable tray322, positioned on the guide rails 324, into and out of the housing 354.

Once secured within the instrument system 350, a depressible manifoldassembly 326 is lowered into contact with the reservoirs, e.g.,reservoirs 304 and 306 in the microfluidic device 302, making sealedcontact between the manifold assembly 326 and the reservoirs 304 and 306by virtue of intervening gasket 314. Depressible manifold assembly 326is actuated and lowered against the microfluidic device 302 throughincorporated servo motor 328 that controls the movement of the manifoldassembly 326, e.g., through a rotating cam (not shown) that ispositioned to push the manifold assembly 326 down against microfluidicdevice 302 and gasket 314, or through another linkage. The manifoldassembly 326 is biased in a raised position by springs 330. Once themanifold assembly 326 is securely interfaced with the reservoirs, e.g.,reservoirs 304 and 306, on the microfluidic device 302, pressures aredelivered to one or more reservoirs, e.g., reservoirs 304 and 306,within each channel network within the microfluidic device 302,depending upon the mode in which the system is operating, e.g., pressureor vacuum drive. The pressures are supplied to the appropriate conduitswithin the manifold 326 from one or both of pumps 332 and 334. Operationof the system is controlled through onboard control processor, shown ascircuit board 356, which is programmed to operate the pumps inaccordance with preprogrammed instructions, e.g., for requisite times orto be responsive to other inputs, e.g., sensors or user inputs. Alsoshown is a user button 338 that is depressed by the user to execute thecontrol of the system, e.g., to extend and retract the tray 322 prior toa run, and to commence a run. Indicator lights 340 are provided toindicate to the user the status of the instrument and/or system run. Theinstrument components are secured to a frame 352 and covered withinhousing 354.

IV. Environmental Control

In addition to flow control components, the systems described herein mayadditionally or alternatively include other interfaced components, suchas environmental control components, monitoring components, and otherintegrated elements.

In some cases, the systems may include environmental control elementsfor controlling parameters in which the channel networks, reagentvessels, and/or product reservoirs are disposed. In many cases, it willbe desirable to maintain controlled temperatures for one or more of thefluidic components or the elements thereof. For example, when employingtransient reactants, it may be desirable to maintain cooler temperaturesto preserve those reagents. Likewise, in many cases partitioning systemsmay operate more optimally at a set temperature, and maintaining thesystem at such temperature will reduce run-to-run variability.Temperature controllers may include any of a variety of differenttemperature control systems, including simple heaters and coolers, fansor radiators, interfaced with the fluidics component portion of thesystem. In preferred aspects, temperature control may be providedthrough a thermoelectric heater/cooler that is directly contacted withthe device, or a thermal conductor that is contacted with the device, inorder to control its temperature. Thermoelectric coolers are widelyavailable and can generally be configured to apply temperature controlto a wide variety of different structures and materials. The temperaturecontrol systems will typically be included along with temperaturesensing systems for monitoring the temperature of the system or keyportions of it, e.g., where the fluidics components are placed, so as toprovide feedback control to the overall temperature control system.

In addition to temperature control, the systems may likewise providecontrol of other environmental characteristics, such as providing acontrolled humidity level within the instrument, and/or providing alight or air sealed environment, e.g., to prevent light damage orpotential contamination from external sources.

V. Monitoring and Detection

The systems described herein also optionally include other monitoringcomponents interfaced with the fluidics components. Such monitoringsystems include, for example, pressure monitoring systems, levelindicator systems, e.g., for monitoring reagent levels withinreservoirs, and optical detection systems, for observing fluids or othermaterials within channels within the fluidics components.

A. Pressure

A variety of different monitoring systems may be included, such aspressure monitoring systems that may allow identification of pluggedchannels, air bubbles, exhaustion of one or more reagents, e.g., thatmay signal the completion of a given operation, or real time feedback offluid flows, e.g., indicating viscosity by virtue of back pressures,etc. Such pressure monitoring systems may often include one or morepressure sensors interfaced with one or more fluidic channels,reservoirs or interfacing components, e.g., within the lines connectingthe pumps to the reservoirs of the device, or integrated into otherconduits coupled to other reservoirs. By way of example, where apositive pressure is applied to multiple inlet reservoirs, pressuresensors coupled to those inlet reservoirs can allow the detection of achannel clog which may be accompanied by a pressure increase, orinjection of air through a channel which may accompany exhaustion of oneor more reagents from a reservoir, which may be accompanied by apressure drop. Likewise, pressure sensors coupled to a reservoir towhich a negative pressure is applied may similarly identifyperturbations in pressure that may be indicative of similar failures oroccurrences. With reference to FIG. 1, pressure sensors may beoptionally integrated into one or more of the lines connecting the pumps118-128 (shown as dashed lines), or integrated directly into thereservoirs 106-116, disposed at the termini of the various channelsegments in the fluidic channel network 104. The sensors incorporatedinto the instrument may typically be operably coupled to the controllerthat is integrated into the instrument, e.g., on circuit board 356 shownin FIG. 3B. Alternatively or additionally, the sensors may be linked,e.g., through appropriate connectors, to an external processor forrecording and monitoring of signals from those sensors.

As will be appreciated, when in normal operation, it would be expectedthat the pressure profiles at the one or more sensors would be expectedto remain relatively steady. However, upon a particular failure event,such as aspiration of air into a channel segment, or a blockage at oneor more channel segments or intersections, would be expected to cause aperturbation in the steady state pressure profiles. For example, for asystem as shown in FIG. 1, that includes an applied negative pressure atan outlet reservoir, e.g., reservoir 116 with an integrated pressuresensor, normal operation of the system would be expected to have arelatively steady state of this negative pressure exhibited at thereservoir. However, in the event of a system disturbance, such asexhaustion of a reagent in one or more of reservoirs 106-114, andresulting aspiration of air into the channels of the system, one wouldexpect to see a reduction in the negative pressure (or an increase inpressure) at the outlet reservoir resulting from the sudden decrease influidic resistance in the channel network resulting from theintroduction of air. By monitoring the pressure profile, the system mayinitiate changes in operation in response to such perturbations,including, e.g., shut down of the pumps, triggering of alarms, or othermeasures, in order to void damaging failure events, e.g., to the systemor the materials being processed therein. As will be appreciated,pressure profiles would be similarly monitorable when using individuallyapplied pressures at multiple reservoirs/channel termini. For example,for positive applied pressures, introduction of air into channels wouldbe expected to cause a drop in pressure at an inlet reservoir, whileclogs or obstructions would be expected to result in increases inpressures at the inlets of a given clogged channel or channels.

In some cases, one or more pressure sensors may be integrated within themanifold or pressure lines that connect to one or more of the reservoirsor other channel termini, as described herein. A variety of pressuresensor types may be integrated into the systems described herein. Forexample, small scale solid state pressure sensors may be coupled,in-line, with pressure or vacuum lines connected to the reservoirs ofthe fluidic components, in order to measure pressure within those linesand at those reservoirs. As with the pumps described herein, pressuresensors may be integrated with one or more of the reservoirs, includingthe outlet and inlet reservoirs, as applicable. In some cases, eachpressure conduit connected to a reservoir within a device may include apressure sensor for monitoring pressures at such reservoirs.

In operation, the pressure sensing system is used to identify pressureperturbations that signal system failures or end-of-run events, such aschannel clogs, air aspiration through channels, e.g., from reagentexhaustion, or the like. In particular, the pressure sensing system isused to trigger system operations when the steady state pressuresmeasured by the pressure sensing system deviate above or below athreshold amount. Upon occurrence of such a perturbation, the system maybe configured to shut down, or reduce applied pressures, or initiateother mitigation measures to avoid adulterating the overall system,e.g., by drawing fluids into the pumping system, or manifold. In certainaspects, the system will be configured to shut down or reduce appliedpressures when the steady state pressure measured in one or more channelsegments deviates from the steady state pressure by more than 10%, morethan 20%, more than 30%, more than 40%, more than 50%, more than 60%, ormore.

In addition to or as an alternative to the pressure sensors describedabove, one or more flow sensors may also be integrated into the system,e.g., within the manifold or flow lines of the system, in order tomonitor flow through the monitored conduit. As with the pressuresensors, these flow sensors may provide indications of excessive flowrates within one or more of the conduits feeding the fluidic device, aswell as provide indications of perturbations in that flow resulting fromsystem problems or fluidics problems, e.g., resulting from channelocclusions or constrictions, exhaustion of one or more fluid reagents,etc.

B. Optical Monitoring and Detection

In addition to pressure sensors, the systems described herein may alsoinclude optical sensors for measurement of a variety of differentparameters within the fluid components of the system, as well as withinother parts of the system. For example, in at least one example, anoptical sensor is positioned within the system such that it is inoptical communication with one or more of the fluid channels in thefluid component. The optical sensor is typically positioned adjacent oneor more channels in the fluid component, so that it is able to detectthe passage of material through the particular channel segment. Thedetection of materials may be by virtue of the change in opticalproperties of the fluids flowing through the channel, e.g., lightscattering, refractive index, or by virtue of the presence of opticallydetectable species, e.g., fluorophores, chromophores, colloidalmaterials, or the like, within the fluid conduits.

In many cases, the optical detection system optionally includes one ormore light sources to direct illumination at the channel segment. Thedirected light may enhance aspects of the detection process, e.g.,providing contrasting light or excitation light in the illumination ofthe contents of the channel. In some cases, the light source may be anexcitation light source for exciting fluorescent components within thechannel segment that will emit fluorescent signals in response. Thesefluorescent signals are then detected by the optical sensor.

FIG. 4 schematically illustrates an example of an optical detectionsystem for monitoring materials within fluidic channels of the fluidicscomponent of the systems described herein. As shown, the opticaldetection system 400 typically includes an optical train 402 placed inoptical communication with one or more channel segments within thefluidic component, e.g., channel segment 404. In particular, opticaltrain 402 is placed within optical communication with channel segment404 in order to optically interrogate the channel segment and/or itscontents, e.g., fluid 406 and particles or droplets 408. Generally, theoptical train will typically include a collection of optical componentsused for conveying the optical signals from the channel segments to anassociated detector or detectors. For example, optical trains mayinclude an objective lens 410 for receiving optical signals from thefluid channel 404, as well as associated optical components, e.g.,lenses 412 and 414, spectral filters and dichroics 416 and 418, andspatial filters, e.g., filter 420, for directing those optical signalsto a detector or sensor 422 (and one or more optional additionalsensors, e.g., sensor 424), such as a CCD or CMOS camera, PMT,photodiode, or other light detecting device.

In some cases, the optical detection system 400 may operate as a lightmicroscope to detect and monitor materials as they pass through thechannel segment(s) in question. In such cases, the optical train 402 mayinclude spatial filters, such as confocal optics, e.g., filter 420, aswell as an associated light source 426, in order to increase contrastfor the materials within the channel segment.

In some cases, the optical detection system may alternatively, oradditionally be configured to operate as a fluorescence detectionmicroscope for monitoring fluorescent or fluorescently labeled materialspassing through the channel segments. In the case of a fluorescencedetection system, light source 426 may be an excitation light source,e.g., configured to illuminate the contents of a channel at a wavelengththat excites fluorescence from the materials within the channel segment.In such cases, the optical train 402, may additionally be configuredwith filter optics to allow the detection of fluorescent emissions fromthe channel without interference from the excitation light source 426.This is typically accomplished through the incorporation of cut-off ornarrow band pass filters, e.g., filter 416 within the optical train tofilter out the excitation wavelength while permitting light of thewavelengths emitted by the fluorescent species to pass and be detected.

In particularly preferred aspects, the optical sensor is providedoptically coupled to one or more of a particle inlet channel segment(through which beads or other particles are injected into thepartitioning region of the fluidic component of the system), e.g.,channel segment 202 of FIG. 2, to monitor the materials being broughtinto the partitioning junction, e.g., monitoring the frequency and flowrates of particles that are to be co-partitioned in the partitioningjunction. Alternatively or additionally, the optical detector may bepositioned in optical communication with the post partitioning channelsegment of the fluidic component, e.g., channel segment 228, to allowthe monitoring of the formed partitions emanating from the partitioningjunction of the fluidic device or structure. In particular, it is highlydesirable to be able to monitor and maintain control of the flow ofparticles that are being introduced into the partitioning region, and tomonitor and control the flow and characteristics of partitions as theyare being generated in order to ensure the proper flow rates andgeneration frequencies for the partitions, as well as to understand theefficiency of the partitioning process.

In a particular example, the optical sensor is used to monitor anddetect partitions as they pass a particular point in the channelsegment. In such cases, the optical sensor may be used to measurephysical characteristics of the partitions, or their components, as theypass the position in the channel, such as the size, shape, speed orfrequency of the partitions as they pass the detector. In other cases,the optical detector or sensor 422 may be configured to detect someother characteristics of the partitions as they pass the detector orsensor 422, e.g., relating to the contents of the partitions.

As noted above, in some cases, the optical detection system will beconfigured to monitor aspects of the contents of the created partitions.For example, in some cases, materials that are to be co-partitioned intoindividual partitions may be monitored to detect the relative ratio ofthe co-partitioned materials. By way of example, two fluid bornematerials, e.g., a reagent, and a bead population, may each bedifferentially optically labeled, and the optical detection system isconfigured to resolve the contribution of these materials in theresulting partitions.

In an example system, two optically resolvable fluorescent dyes may beseparately suspended into each of the first reagent and the secondreagents that are to be co-partitioned. The relative ratio of the firstand second reagents in the resulting partition will be ascertainable bydetecting the fluorescent signals associated with each fluorescent dyein the resulting partition. Accordingly, the optical detection systemwill typically be configured for at least two-color fluorescent optics.Such two color systems typically include one or more light sources thatprovide excitation light at the appropriate wavelengths to excite thedifferent fluorescent dyes in the channel segment. These systems alsotypically include optical trains that differentially direct thefluorescent emissions from those dyes to different optical detectors orregions on the same detector. With reference to FIG. 4, for example, twooptically distinguishable fluorescent dyes may be co-partitioned intodroplets, e.g., droplets 408 within channel segment 404. Upon excitationof those fluorescent dyes by light source 426, two optically resolvablefluorescent signals are emitted from the droplets 408, shown as solidarrow 428. The mixed fluorescent signals, along with transientexcitation light are collected through objective 410 and passed throughoptical train 402. Excitation light is filtered out of the signal pathby inclusion of an appropriate filter, e.g., filter 416, which mayinclude one or more cut-off or notch filters that pass the fluorescentlight wavelengths while rejecting the excitation wavelengths. The mixedfluorescent signals are then directed toward dichroic mirror 420, whichallows one of the fluorescent signals (shown by arrow 430) to passthrough to a first detector 422, while reflecting a second, spectrallydifferent fluorescent signal (shown by arrow 432), to second detector424.

The intensities of each fluorescent signals associated with each dye,are reflective of the concentration of those dyes within the droplets.As such, by comparing the ratio of the signal from each fluorescent dye,one can determine the relative ratio of the first and second fluidswithin the partition. Further, by comparing the detected fluorescence toknown extinction coefficients for the fluorescent dyes, as well as thesize of observed region, e.g., a droplet, one can determine theconcentration of each component within a droplet. As will beappreciated, where looking to partition particle based reagents intodroplets, when using a fluorescently labeled particle, these systemsalso will allow one to ascertain the relative number of particles withina partition, as well as identifying partitions that contain noparticles.

In other aspects, the optical detection systems may be used to determineother characteristics of the materials, particles, partitions or thelike, flowing through the channel segments, including, for example,droplet or particle size, shape, flow rate, flow frequency, and othercharacteristics. In at least one aspect, optical detectors provided areconfigured to better measure these characteristics. In one aspect, thisis achieved through the incorporation of a line scan camera or detector,e.g., camera 510, into the optical system, that images across a channelsegment in a detection line in order to process images of the materialsas they pass through the detection line. This is schematicallyillustrated in FIG. 5, top panel. As shown, a channel segment 502 isprovided wherein materials, and particularly particulate or dropletbased materials are being transported. The optical detection systemimages a line across the channel segment 502 (indicated as image zone504). Because the line scan camera employs a line scanner, rather than atwo-dimensional array of pixels associated with other camera types, itresults in substantially less image processing complexity, allowinggreater flexibility of operation.

In addition to using a line scan camera system, in some cases, it isdesirable to provide higher resolution imaging using such camera systemsby angling the detection line across the channel segment 502, as shownin FIG. 5, bottom panel. In particular, assuming a linear,one-dimensional array of pixels in a line scan camera (schematicallyillustrated as pixels 506 in camera 508), one would expect an image thatis reflective of those pixels (schematically illustrated as image 510).Typically, the angle θ at which the detection line (indicated as imagezone 504) is angled across the channel segment 502 will range from about5-80 degrees from an axis Y perpendicular to the channel segment 502,more specifically 15-75 degrees, 20-70 degrees, 25-65 degrees, 30-60degrees, 35-55 degrees, 40-50 degrees, or in some cases approximately 45degrees. Though recited in terms of certain ranges, it will beunderstood that all ranges from the lowest of the lower limits to thehighest of the upper limits are included, including all intermediateranges or specific angles, within this full range or any specificallyrecited range. By angling the camera and the detection line/image zone504, one achieves an effective closer spacing of the pixels as theyimage flowing materials. The resulting image thus is of higherresolution across the channel, as shown by image 512, than for theperpendicularly oriented image zone, as shown by image 510. By providinghigher resolution, one is able to obtain higher quality images of theparticles, droplets or other materials flowing through the channelsegments of the device, and from that, derive the shape, size and othercharacteristics of these materials.

As will be appreciated, as the optical detection systems may be used tomonitor flow rates within channel segments of a device, these detectionsystems may, as with the pressure monitoring systems described above,identify perturbations in the operation of the system. For example,where a reagent well is exhausted, allowing air to be passed through thechannels of the device, while leading to a pressure drop across therelevant channel segments, it will also result in an increase in flowrate through that channel segment resulting from the lower fluidicresistance in that channel. Likewise, an obstructed channel segment willin many cases, lead to a reduced flow rate in downstream channelsegments connected to the obstructed channel segment. As such,perturbations in flow rates measured optically, may be used to indicatesystem failures or run completions or the like. In general,perturbations of at least 5% in the optically determined flow rate, atleast 10%, at least 20%, at least 30%, at least 40%, or at least 50%,will be indicative of a problem during a processing run, and may resultin a system adjustment, shutdown or the like.

FIG. 8 illustrates optical monitoring processes and systems as describedherein for use in identifying perturbations in flow within channels of afluidic network. As shown, a single a microfluidic device, e.g., asshown in FIG. 2, is run under applied pressures at each of the variousinlet reservoirs, e.g., reservoirs 230, 232 and 234, under constantpressure. The flow rate of droplets is measured within an outlet channelsegment, e.g., channel segment 228 using an optical imaging system. Theflow rate of a normally operating channel segment is plotted in thefirst portion 302 of the flow rate plot shown in FIG. 8. Upon exhaustionof one reagent, e.g., the oil in reservoir 234, air is introduced intothe channel network, resulting in a reduced fluidic resistance, causingan increase in the flow rate, as shown in the second portion 304 of theplot.

VI. Reagent Detection

In addition to the components described above, in some cases, theoverall systems described herein may include additional componentsintegrated into the system, such components used to detect the presenceand amount of reagents present in any reagent vessel component of thesystem, e.g., in a reservoir of a microfluidic device, an amplificationtube, or the like. A variety of components may be used to detect thepresence and/or amount of reagents in any vessel, including, forexample, optical detection systems, that could include lighttransmission detectors that measure whether light is altered in passingthrough a reservoir based upon presence of a fluid, or machine visionsystems that image the reservoirs and determine whether there is fluidin the reservoir and even the level of fluid therein. Such detectionsystems would be placed in optical communication with the reservoirs orother vessels of the system. In other cases, electrical systems may beused that insert electrodes into a reservoir and measure changes incurrent flow through those electrodes based upon the presence or absenceof fluid within the reservoir or vessel.

VII. Additional Sensors/Monitoring

In addition to the sensing systems described above, a number ofadditional sensing systems may also be integrated into the overallsystems described herein. For example, in some cases, the instrumentsystems may incorporate bar-code reader systems in one or morefunctional zones of the system. For example, in some cases, a barcodereader may be provided adjacent a stage for receiving one or more sampleplates, in order to record the identity of the sample plat and correlateit to sample information for that plate. Likewise, barcode readers maybe positioned adjacent a microfluidic device stage in a partitioningzone, in order to record the type of microfluidic device being placed onthe stage, as reflected by a particular barcode placed on the device. Bybarcoding and reading the specific device, one could coordinate thespecifics of an instrument run that may be tailored for different devicetypes. A wide variety of barcode types and readers are generally used inresearch instrumentation, including both one dimensional and twodimensional barcode systems.

Other detection systems that are optionally integrated into the systemsdescribed herein include sensors for the presence or absence ofconsumable components, such as microfluidic devices, sample plates,sample tubes, reagent tubes or the like. Typically, these sensor systemsmay rely on one or more of optical detectors, e.g., to sense thepresence or absence of a physical component, such as a plate, tube,secondary holder, microfluidic chip, gasket, etc., or mechanicalsensors, e.g., that are actuated by the presence or absence of a plate,microfluidic device, secondary holder, tube, gasket, etc. These sensorsystems may be integrated into one or more tube slots or wells, platestages or microfluidic device stages. In the event a particularcomponent is missing, the system may be programmed to provide an alertor notification as well as optionally or additionally preventing thestart of a system run or unit operation.

II. Integrated Workflow Processes

The instrument systems described above may exist as standaloneinstruments, or they may be directly integrated with other systems orsubsystems used in the particular workflow for the application for whichthe partitioning systems are being used. As used herein, integration ofsystems and subsystems denotes the direct connection or joining of thesystems and/their respective processes into an integrated system orinstrument architecture that does not require user intervention inmoving a processed sample or material from a first subsystem to a secondsubsystem. Typically, such integration denotes two subsystems that arelinked into a common architecture, and include functional interactionsbetween those subsystems, or another subsystem common to both. By way ofexample, such interconnection includes exchange of fluid materials fromone subsystem to another, exchange of components, e.g., plates, tubes,wells, microfluidic devices, etc., between two subsystems, andadditionally, may include integrated control components betweensubsystems, e.g., where subsystems are controlled by a common processor,or share other common control elements, e.g., environment control, fluidtransport systems, etc.

For ease of discussion, these integrated systems are described withrespect to the example of nucleic acid applications. In this example,the partitioning instrument systems may be integrated directly with oneor more sample preparation systems or subsystems that are to be usedeither or both of upstream and/or downstream in the specific overallworkflow. Such systems may include, for example, upstream processsystems or subsystems, such as those used for nucleic acid extraction,nucleic acid purification, and nucleic acid fragmentation, as well asdownstream processing systems, such as those used for nucleic acidamplification, nucleic acid purification and nucleic acid sequencing orother analyses.

For purposes of illustration, the integration of the partitioningprocess components described above, with upstream and/or downstreamprocess workflow components is illustrated with respect to a preferredexemplary nucleic acid sequencing workflow. In particular, thepartitioning systems described herein are fluidly and/or mechanicallyintegrated with other systems utilized in a nucleic acid sequencingworkflow, e.g., amplification systems, nucleic acid purificationsystems, cell extraction systems, nucleic acid sequencing systems, andthe like.

FIG. 6 schematically illustrates an exemplary process workflow forsequencing nucleic acids from sample materials and assembling theobtained sequences into whole genome sequences, contig sequences, orsequences of significantly large portions of such genomes, e.g.,fragments of 10 kb or greater, 20 kb or greater, 50 kb or greater, or100 kb or greater, exomes, or other specific targeted portions of thegenome(s).

As shown, a sample material, e.g., comprising a tissue or cell sample,is first subjected to an extraction process 602 to extract the genomicor other nucleic acids from the cells in the sample. A variety ofdifferent extraction methods are commercially available and may varydepending upon the type of sample from which the nucleic acids are beingextracted, the type of nucleic acids being extracted, and the like. Theextracted nucleic acids are then subjected to a purification process604, to remove extraneous and potentially interfering sample componentsfrom the extract, e.g., cellular debris, proteins, etc. The purifiednucleic acids may then be subjected to a fragmentation step 606 in orderto generate fragments that are more manageable in the context of thepartitioning system, as well as optional size selection step, e.g.,using a SPRI bead clean up and size selection process.

Following fragmentation, the sample nucleic acids may be introduced intothe partitioning system, which is used to generate the sequenceablelibrary of nucleic acid fragments. Within the partitioning system largersample DNA fragments are co-partitioned at step 608, along with barcodedprimer sequences, such that each partition includes a particular set ofprimers representing a single barcode sequence. Additional reagents mayalso be co-partitioned along with the sample material, including, e.g.,release reagents for releasing the primer/barcode oligonucleotides fromthe beads, DNA polymerase enzyme, dNTPs, divalent metal ions, e.g.,Mg2+, Mn2+, and other reagents used in carrying out primer extensionreactions within the partitions. These released primers/barcodes arethen used to generate a set of barcoded overlapping smaller fragments ofthe larger sample nucleic acid fragments at amplification step 610,where the smaller fragments include the barcode sequence, as well as oneor more additional sequencing primer sequences.

Following generation of the sequencing library, additional process stepsmay be carried out prior to introducing the library onto a sequencersystem. For example, as shown, the barcoded fragments may be taken outof their respective partitions, e.g., by breaking the emulsion, and besubjected to a further amplification process at step 612 where thesequenceable fragments are amplified using, e.g., a PCR based process.Either within this process step or as a separate process step, theamplified overlapping barcoded fragments may have additional sequencesappended to them, such as reverse read sequencing primers, sample indexsequences, e.g., that provide an identifier for the particular samplefrom which the sequencing library was created.

In addition, either after the amplification step (as shown) or prior tothe amplification step, the overlapping fragment set may be sizeselected, e.g., at step 614, in order to provide fragments that arewithin a size nucleotide sequence length range that is sequenceable bythe sequencing system being used. A final purification step 616 may beoptionally performed to yield a sequenceable library devoid ofextraneous reagents, e.g., enzymes, primers, salts and other reagents,that might interfere with or otherwise co-opt sequencing capacity of thesequencing system. The sequencing library of overlapping barcodedfragments is then run on a sequencing system at step 618 to obtain thesequence of the various overlapping fragments and their associatedbarcode sequences.

In accordance with the instant disclosure, it will be appreciated thatthe steps represented by the partitioning system, e.g., step 606, may bereadily integrated into a unified system with any one or more of any ofsteps 602-606 and 610-618. This integration may include integration onthe subsystem level, e.g., incorporation of adjacent processing systemswithin a unified system architecture. Additionally or alternatively, oneor more of these integrated systems or components thereof, may beintegrated at the component level, e.g., within one or more individualstructural components of the partitioning subsystem, e.g., in anintegrated microfluidic partitioning component.

As used herein, integration may include a variety of types ofintegration, including for example, fluidic integration, mechanicalintegration, control integration, electronic or computationalintegration, or any combination of these. In particularly preferredaspects, the partitioning instrument systems are fluidly and/ormechanically integrated with one or more additional upstream and/ordownstream processing subsystems.

A. Fluidic Integration

In the case of fluidic integration, it will be understood that suchintegration will generally include fluid transfer components fortransferring fluid components to or from the inlets and outlets, e.g.,the reservoirs, of the fluidic component of the partitioning system.These fluid transfer components may include any of a variety ofdifferent fluid transfer systems, including, for example, automatedpipetting systems that access and pipette fluids to or from reservoirson the fluidic component to transfer such fluids to or from reservoirs,tubes, wells or other vessels in upstream or downstream subsystems. Suchpipetting systems may typically be provided in the context ofappropriate robotics within an overall system architecture, e.g., thatmove one or both of the fluidics component and/or the pipetting systemrelative to each other and relative to the originating or receivingreservoir, etc. Alternatively, such systems may include fluidic conduitsthat move fluids among the various subsystem components. Typically, hardwired fluidic conduits are reserved for common reagents, buffers, andthe like, and not used for sample components, as they would be subjectto sample cross contamination.

In one example, a fluid transfer system is provided for transferring oneor more fluids into the reservoirs that are connected to the channelnetwork of the fluidics component. For example, in some cases, fluids,such as partitioning oils, buffers, reagents, e.g., barcode beads orother reagents for a particular application, may be stored in discretevessels, e.g., bottles, flasks, tubes or the like, within the overallsystem. These storage vessels would optionally be subject toenvironmental control aspects as well, to preserve their efficacy, e.g.,refrigeration, low light or no light environments, etc.

Upon commencement of a system run, those reagent fluids would betransported to the reservoirs of a fluidic component, e.g., amicrofluidic device, that was inserted into the overall system. Again,reagent transport systems for achieving this may include dispensingsystems, e.g., with pipettors or dispensing tubes positioned orpositionable over the reservoirs of the inserted device, and which areconnected to the reagent storage vessels and include pumping systems.

Likewise, fluid transport systems may also be included to transfer thepartitioned reagents from the outlet of the fluidic component, e.g.,reservoir 238 in FIG. 2, and transported to separate locations withinthe overall system for subsequent processing, e.g., amplification,purification etc.

In other cases, the partitions may be maintained within the outletreservoir of the fluidic component, which is then directly subjected tothe amplification process, e.g., through a thermal controller placedinto thermal contact with the outlet reservoir, that can perform thermalcycling of the reservoir's contents. This thermal controller may be acomponent of the mounting surface upon which the microfluidic device ispositioned, or it may be a separate component that is brought intothermal communication with the microfluidic device or the reservoir.

However, in some cases, fully integrated systems may be employed, e.g.,where the transfer conduits pass the reagents through thermally cycledzones to effect amplification. Likewise, alternative fluid transfersystems may rely upon the piercing of a bottom surface of a reservoir ona given device to allow draining of the partitions into a subsequentreceptacle for amplification.

B. Mechanical Integration

In cases of mechanical integration, it will be understood that suchintegration will generally include automated or automatable systems forphysically moving system components, such as sample plates, microfluidicdevices, tubes, vials, containers, or the like, from one subsystem toanother subsystem. Typically, these integrated systems will be containedwithin a single unified structure, such as a single casing or housing,in order to control the environments to which the various process steps,carried out by the different system components, are exposed. In somecases, different subsystem components of the overall system may besegregated from other components, in order to provide differentenvironments for different unit operations performed within theintegrated system. In such cases, pass-throughs may be provided withclosures or other movable barriers to maintain environmental control asbetween subsystem components.

Mechanical integration systems may include robotic systems for movingsample containing components from one station to another station withinthe integrated system. For example, robotic systems may be employedwithin the integrated system to move lift and move plates from onestation in a first subsystem, to another station in another subsystem.

Other mechanical integration systems may include conveyor systems, rotortable systems, inversion systems, or other translocation systems thatmove, e.g., a partitioning microfluidic device, tubes, or multiwallplate or plates, from one station to another station within the unifiedsystem architecture, e.g., moving a microfluidic device from its controlstation where partitions are generated to a subsequent processingstation, such as an amplification station or fluid transfer station.

C. Examples of Integration

A number of more specific simple examples of integration of theaforementioned process components are described below.

In some cases, the up front process steps of sample extraction andpurification may be integrated into the systems described herein,allowing users to input tissue, cell, or other unprocessed samples intothe system in order to yield sequence data for those samples. Suchsystems would typically employ integrated systems for lysis of cellmaterials and purification of desired materials from non-desiredmaterials, e.g., using integrated filter components, e.g., integratedinto a sample vessel that could be integrated onto a microfluidic deviceinlet reservoir following extraction and purification. These systemsagain would be driven by one or more of pressure or vacuum, or in somecases, by gravitation al flow or through centrifugal driving, e.g.,where sample vessels are positioned onto a rotor to drive fluidmovements.

In some cases, it may be desirable to have sample nucleic acidssize-selected, in order to better optimize an overall sample preparationprocess. In particular, it may be desirable to have one or more selectedstarting fragment size ranges for nucleic acid fragments that are to bepartitioned, fragmented and barcoded, prior to subjecting thesematerials to sequencing. This is particularly useful in the context ofpartition-based barcoding and amplification where larger startingfragment sizes may be more desirable. Examples of available sizeselection systems include, e.g., the Blue Pippen® system, available fromSage Sciences (See also U.S. Pat. No. 8,361,299), that relies upon sizeseparation through an electrophoretic gel system, to provide relativelytightly defined fragment sizes.

In accordance with the present disclosure, systems may include anintegrated size selection system for generating nucleic acid fragmentsof selected sizes. While in some cases, these size selection componentsmay be integrated through fluid transport systems that transportfragments into the inlet reservoirs of the fluidic components, e.g.,pipetting systems, in certain cases, the size selection system may beintegrated within the fluidic component itself, such that samples ofvaried fragment sizes may be input into the device by the user, followedby an integrated size separation process whereby selected fragment sizesmay be allocated into inlet reservoirs for the fluidic components of thedevice.

For example, and as shown in FIG. 7, a size selection component 700including a capillary or separation lane 702, is integrated into amicrofluidic device. An electrophoretic controller is coupled to theseparation lane via electrodes 704, 706 and 708 that apply a voltagedifferential across the separation matrix in lane 702 in order to drivethe size-based separation of nucleic acid samples that are introducedinto well 710. In operation, a separation voltage differential isapplied across the separation lane by applying the voltage differentialbetween sample reservoir 710 and waste reservoir 712. At the point inthe separation at which the desired fragment size enters into junction714, the voltage differential is applied between reservoir 710 andelution reservoir 716, by actuation of switch 718. This switch of theapplied voltage differential then drives the desired fragment size intothe elution reservoir 716, which also doubles as the sample inletreservoir for the microfluidic device, e.g., reservoir 232 in FIG. 2.Once sufficient time has passed for direction of the desired fragmentinto reservoir 716, the voltage may again be switched as betweenreservoir 710 and waste reservoir 712.

Upon completion of the separation, fragments that have been driven intothe sample elution reservoir/sample inlet reservoir, may then beintroduced into their respective microfluidic partitioning channelnetwork, e.g., channel network 720, for allocation into partitions forsubsequent processing. As will be appreciated, in cases where anelectrophoretic separation component is included within the system,e.g., whether integrated into the microfluidic device component orseparate from it, the systems described herein will optionally includean electrophoretic controller system that delivers appropriate voltagedifferentials to the associated electrodes that are positioned inelectrical contact with the content of the relevant reservoirs. Suchsystems will typically include current or voltage sources, along withcontrollers for delivering desired voltages to specified electrodes atdesired times, as well as actuation of integrated switches. Thesecontroller systems, either alone, or as a component of the overallsystem controller, will typically include the appropriate programming toapply voltages and activate switches to drive electrophoresis of samplefragments in accordance with a desired profile.

As will be appreciated, a single microfluidic device may includemultiple partitioning channel networks, and as such, may also includemultiple size separation components integrated therein as well. Thesesize separation components may drive a similar or identical sizeseparation process in each of the different components, e.g., to providethe same or similar sized fragments to each different partitioningchannel network. Alternatively, the different size separation componentsmay drive a different size selection, e.g., to provide different sizedfragments to the different partitioning networks. This may be achievedthrough the inclusion of gel matrices having different porosity, e.g.,to affect different separation profiles, or it may be achieved byproviding different voltage profiles or switching profiles to theelectrophoretic drivers of the system.

As will be appreciated, for microfluidic devices that include multipleparallel arranged partitioning channel networks, multiple separationchannels may be provided; each coupled at an elution zone or reservoirthat operates as or is coupled to a different inlet reservoir for thepartition generating fluidic network. In operation, a plurality ofdifferent separation channel components maybe provided integrated into amicrofluidic device. The separation channels again are mated with orinclude associated electrodes for driving electrophoresis of nucleicacids or other macromolecular sample components, through a gel matrixwithin the separation channels. Each of the different separationchannels may be configured to provide the same or differing levels ofseparation, e.g., resulting in larger or smaller eluted fragments intothe elution zone/inlet reservoir of each of the different partitioningchannel networks. In cases where the separation channels providedifferent separation, each of the different channel networks would beused to partition sample fragments of a selected different size, withthe resulting partitioned fragments being recovered for each channelnetwork in a different outlet or recovery reservoir, respectively.

3. Amplification

In some cases, the systems include integration of one or more of theamplification process components, e.g., steps 610 and 612, into theoverall instrument system. In particular, as will be appreciated, thisintegration may be as simple as incorporating a temperature controlsystem within thermal communication with the product reservoir on thefluidic component of the system, e.g., reservoir 238 in FIG. 2, suchthat the contents of the reservoir may be thermally cycled to allowpriming, extension, melting and re-priming of the sample nucleic acidswithin the partitions by the primer/barcode oligonucleotides in order tocreate the overlapping primer sequences template off of the originalsample fragment. Again, such temperature control systems may includeheating elements thermally coupled to a portion of the fluidic componentso as to thermally cycle the contents of the outlet reservoir.

Alternatively, the integration of the amplification system may providefor fluid transfer from the outlet reservoir of the fluidic component toan amplification reservoir that is positioned in thermal contact withthe above described temperature control system, e.g., in a temperaturecontrolled thermal cycler block, within the instrument, that iscontrolled to provide the desired thermal cycling profile to thecontents taken from the outlet reservoir. As described above, this fluidtransfer system may include, e.g., a pipetting system for drawing thepartitioned components out of the outlet reservoir of the microfluidicdevice and depositing them into a separate reservoir, e.g., in a well ofa multiwall plate, or the like. In another alternative configuration,fluid transfer between the microfluidic device and the amplificationreservoir may be directed by gravity or pressure driven flow that isactuated by piercing a lower barrier to the outlet reservoir of themicrofluidic device, allowing the generated partitions to drain or flowinto a separate reservoir below the microfluidic device that is inthermal communication with a temperature control system that operates tothermally cycle the resultant partitions through desired amplificationthermal profiles.

In a particular example, and with reference to the nucleic acid analysisworkflow set forth above, the generated partitions from step 608 may beremoved from the fluidics component by an integrated fluid transfersystem, e.g., pipettors, that withdraw the created partitions form,e.g., reservoir 238 of FIG. 2, and transport those partitions to anintegrated thermal cycling system in order to conduct an amplificationreaction on the materials contained within those partitions. Typically,the reagents necessary for this initial amplification reaction (shown atstep 610, in FIG. 6), will be co-partitioned in the partitions. In manycases, the integrated thermal cycling systems may comprise separatereagent tubes disposed within thermal cycling blocks within theinstrument, in order to prevent sample to sample cross contamination. Insuch cases, the fluid transport systems will withdraw the partitionedmaterials from the outlet reservoir and dispense them into the tubesassociated with the amplification system.

4. Size Selection of Amplification Products

Following amplification and barcoding step 610, the partitioned reagentsare then pooled by breaking the emulsion, and subjected to additionalprocessing. Again, this may be handled through integrated fluid transfersystems that may introduce reagents into the wells or tubes in which thesample materials are contained, or by transferring those components toother tubes in which such additional reagents are located. In somecases, mechanical components may also be included within the system toassist in breaking emulsions, e.g., through vortexing of sample vessels,plates, or the like. Such vortexing may again be provided within a setstation within the integrated system. In some cases, this additionalprocessing may include a size selection step in order to providesequenceable fragments of a desired length.

5. Additional Processing and Sequencing

Following further amplification, it may be desirable to includeadditional clean up steps to remove any unwanted proteins or othermaterials that may interfere with a sequencing operation. In such cases,solid phase DNA separation techniques are particularly useful,including, the use of nucleic acid affinity beads, such as SPRI beads,e.g., Ampure® beads available from Beckman-Coulter, for purification ofnucleic acids away from other components in fluid mixtures. Again, aswith any of the various unit operations described herein, this step maybe automated and integrated within the overall integrated instrumentsystem.

In addition to integration of the various upstream processes ofsequencing within an integrated system, in some cases, these integratedsystems may also include an integrated sequencer system. In particular,in some cases, a single integrated system may include one, two, three ormore of the unit process subsystems described above, integrated with asequencing subsystem, whereby prepared sequencing libraries may beautomatically transferred to the sequencing system for sequenceanalysis. In such cases, following a final pre-sequencing process, theprepared sequencing library may be transferred by an integrated fluidtransfer system, to the sample inlet of a sequencing flow cell or othersequencing interface. The sequencing flow cell is then processed in thesame manner as non-integrated sequencing samples, but without userintervention between library preparation and sequencing.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. For example, particle delivery canbe practiced with array well sizing methods as described. Allpublications, patents, patent applications, and/or other documents citedin this application are incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application, and/or other document were individually andseparately indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. A device configured to facilitate sampleprocessing, comprising: a holder configured to receive a microfluidicdevice, wherein said holder is coupled to a rotatable body, wherein saidrotatable body is configured to alternate between a closed configurationand an open configuration, wherein said microfluidic device comprises aplurality of channel networks for partitioning a sample into a pluralityof partitioned samples, said plurality of channel networks beingconnected to a plurality of inlet and outlet reservoirs, and wherein (i)in said closed configuration, said holder is configured to allowpressure communication between said plurality of inlet and outletreservoirs and an interfaced instrument, and (ii) in said openconfiguration, said holder is configured to permit said microfluidicdevice to be inserted into or removed from said holder.
 2. The device ofclaim 1, further comprising a gasket coupled to said rotatable body. 3.The device of claim 2, wherein said gasket is removable from saidrotatable body.
 4. The device of claim 2, wherein said gasket comprisessecuring features configured to mate with complementary features on saidrotatable body.
 5. The device of claim 2, wherein said gasket comprisesa plurality of apertures configured to be aligned with an inletreservoir or an outlet reservoir of a channel network of said pluralityof channel networks when said microfluidic device is secured in saidholder and said rotatable body is in said closed configuration.
 6. Thedevice of claim 1, wherein said plurality of channel networks aresubstantially parallel to one another when said microfluidic device issecured in said holder.
 7. The device of claim 1, wherein a gasket iscoupled to said microfluidic device.
 8. The device of claim 7, whereinsaid gasket is removable from said microfluidic device.
 9. The device ofclaim 1, wherein a channel network of said plurality of channel networkscomprises a plurality of interconnected fluid channels connected at achannel junction, wherein said channel junction is configured to combinea first fluid containing said sample with a stream of a second fluidimmiscible with said first fluid, to partition said sample into discretedroplets within said second fluid, to thereby provide said partitionedsamples within said discrete droplets, which discrete droplets arestored in an outlet reservoir of said plurality of outlet reservoirs ora storage vessel.
 10. The device of claim 9, wherein said plurality ofinterconnected fluid channels is part of a monolithic microfluidicstructure having intersecting fluid channels.
 11. The device of claim 1,wherein in said open configuration, said rotatable body is configured tosupport said holder against a surface.
 12. The device of claim 1,further comprising at least one monitoring component configured tointerface with a channel network of said plurality of channel networkswhen said microfluidic device is received in said holder, wherein saidat least one monitoring component is configured to observe or monitorone or more characteristics or properties of said at least one of saidplurality of channel networks and fluids flowing therein.
 13. The deviceof claim 12, wherein a monitoring component of said at least onemonitoring component is selected from the group consisting of atemperature sensor, a pressure sensor, and a humidity sensor.
 14. Thedevice of claim 1, wherein at least one channel of said channel networkcomprises a channel segment that widens, wherein said channel segment isconfigured to control flow by breaking capillary forces acting to draw afluid into said at least one channel.
 15. The device of claim 1, whereinat least one channel of said channel network comprises a passive checkvalve.
 16. The device of claim 1, wherein a channel network of saidplurality of channel networks comprises: a first channel segment fluidlyconnected to a source of barcode reagents; a second channel segmentfluidly connected to a source of said sample, wherein said first channelsegment and said second channel segment are fluidly connected to a firstchannel junction; a third channel segment and a fourth channel segment,wherein said third channel segment is fluidly connected to said firstchannel junction, wherein said fourth channel segment is fluidlyconnected to a source of partitioning fluid, and wherein said thirdchannel segment and said fourth channel segment are fluidly connected toa second channel junction; and a fifth channel segment fluidly connectedto said second channel junction, wherein said instrument is configuredto interface with said channel network to (i) drive flow of said barcodereagents and said sample into said first channel junction to form areagent mixture comprising said barcode reagents and said sample in saidthird channel segment, and (ii) drive flow of said reagent mixture andsaid partitioning fluid into said second channel junction to formdroplets comprising said reagent mixture in a stream of partitioningfluid within said fifth channel segment.
 17. The device of claim 1,wherein in said open configuration, said holder is configured to allowsaid microfluidic device to stand at a desired angle to facilitaterecovery of a partitioned sample of said plurality of partitionedsamples from an outlet reservoir of said plurality of inlet and outletreservoirs.
 18. The device of claim 17, wherein said desired angle is anangle in a range from 20 degrees to 70 degrees.
 19. The device of claim18, wherein said desired angle is in a range from 40 degrees to about 50degrees.
 20. The device of claim 19, wherein said desired angle is about45 degrees.
 21. The device of claim 1, wherein said instrumentcomprises: (a) a retractable tray supporting and seating said holder,and slideable into and out of said instrument; (b) a depressiblemanifold assembly configured to be actuated and lowered to themicrofluidic device and to sealaby interface with the plurality of inletand outlet reservoirs, when said microfluidic device is received in saidholder; (c) at least one fluid drive component configured to apply apressure differential between the plurality of inlet and outletreservoirs when said microfluidic device is received in said holder; and(d) a controller configured to operate at least one fluid drivecomponent to apply said pressure differential depending on a mode ofoperation or according to preprogrammed instructions.